Characterization of pluripotent stem cells

Abstract

Characterization of pluripotent stem cells is required for the registration of stem cell lines and allows for an impartial and objective comparison of the results obtained when generating multiple lines. It is therefore crucial to establish specific, fast and reliable protocols to detect the hallmarks of pluripotency. Such protocols should include immunocytochemistry (takes 2 d), identification of the three germ layers in in vitro-derived embryoid bodies by immunocytochemistry (immunodetection takes 3 d) and detection of differentiation markers in in vivo-generated teratomas by immunohistochemistry (differentiation marker detection takes 4 d). Standardization of the immunodetection protocols used ensures minimum variations owing to the source, the animal species, the endogenous fluorescence or the inability to collect large amounts of cells, thereby yielding results as fast as possible without loss of quality. This protocol provides a description of all the immunodetection procedures necessary to characterize mouse and human stem cell lines in different circumstances.

Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

“…However, in most cases, a definite verification of gene expression is impossible on living cells. The detection of endogenous pluripotency markers such as OCT4, SOX2, NANOG, GDF3, and REX1 requires fixation of the cells prior to staining [16][17][18] or is accomplished only after lysis by an RT-PCR analysis [17,18]. With such a manipulation, the cells are lost for further culture.…”

“…This is feasible and routinely performed for cell membrane-based antigens such as TRA-1-60 or SSEA4 [18] although the antibodies might nevertheless perturb the cells [19] and these "early pluripotency markers" do not allow discrimination of terminally stable reprogrammed iPS cells. Also for alkaline phosphatase (AP), which is another early pluripotency marker mostly assessed on fixed cells [16], a fluorescent reporter dye ("AP Live Stain") has been lately developed for live screening [20]. In addition, a fluorescent small molecule named CDy1 has been reported to specifically stain living embryonic stem cells (ESCs) and iPS cells [21].…”

Good Cell Culture Practice for stem cells and stem-cell-derived models

“…Low levels of aneuploidy in a diploid culture may be quite normal as this appears commonly in hESC and iPSC. Marti et al, 2013;Singh et al, 2012;Brivanlou et al, 2003;Gonzalez et al, 2011Andrews, 2002Draper et al, 2002;Henderson et al, 2002;Pera et al, 2000Sperger et al, 2003Richards et al, 2004Nazareth et al, 2013 Itskovitz-Eldor et al, 2000 Chambers et al, 2009;Borowiak et al, 2009;Burridge et al, 2012;Kattman et al, 2011Gertow et al, 2007Gropp et al, 2012Avior et al, 2015Muller et al, 2011Bock et al, 2011Cahan et al, 2014a ture, the quality of the passage method, and the split ratio. It is important to minimize the passage level of cells in routine use and to replace the in-use stock from a frozen cell bank on a regular basis.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…The limitless self-renewal and differentiation properties of stem cells enable them to produce the large quantities of specific cell types for basic research and drug discovery in regenerative therapies [108], especially for neurodegenerative diseases [109], such as Alzheimer's, Parkinson's, and Huntington's diseases. Stem cells have been widely characterized by their biochemical properties via various molecular methods, such as immunocytochemistry, immunohistochemistry, transcriptional expression profiling, and single-cell RNA sequencing [110], [111]. In the past decade, researchers have begun to evaluate the influence of physical cues on stem cells [85].…”